Chronic viral hepatitis and its long-term sequelae, cirrhosis and HCC, represent a major global health problem. Considerable progress has been made in the control and treatment of chronic viral hepatitis, but the success is still limited, and further progress will depend on a more thorough knowledge of the molecular mechanisms of pathogenesis. The chimpanzee model has been fundamental for the discovery of the etiologic agents and for understanding the diseases caused by the hepatitis viruses, but it is not a suitable model for fulminant hepatitis and liver fibrogenesis. To investigate the pathogenesis of liver diseases in humans, we started a collaboration with the Liver Transplanation Center and the Liver Unit of the University of Cagliari, Italy, which is an invaluable source of clinical samples. This collection of specimens serves as the basis for the translational research that I have initiated at the NIH since I joined the LID as a senior investigator in 2007. Our main research strategy is to combine the molecular analysis with the clinical, virologic and histopathologic data. 1. Molecular mechanisms of pathogenesis of acute liver failure (ALF) Hepatitis B virus (HBV)-associated ALF is a dramatic clinical syndrome due to a sudden loss of hepatic cells leading to multiorgan failure. While liver damage in classic acute hepatitis B is T-cell mediated, the pathogenesis of HBV-associated ALF is unknown. Access to liver tissue collected at the time of liver transplantation from two well-defined cases of HBV-associated ALF provided a unique opportunity to establish a molecular definition of this disease (Farci et al. PNAS 2010). By gene expression analysis, we demonstrated that ALF is characterized by an overwhelming B-cell signature centered in the liver with massive accumulation of plasma cells secreting IgG and IgM, accompanied by complement deposition. By phage-display libraries, we discovered that the molecular target of these antibodies is the hepatitis B core antigen (HBcAg);that these antibodies display a restricted variable heavy chain (VH) repertoire and lack somatic mutations;and that unrelated individuals with ALF use an identical predominant VH gene with unmutated variable domain (VH1-3) for both IgG and IgM anti-HBc antibodies, indicating that HBcAg is the target of a germline human VH gene. Thus, in contrast to acute hepatitis B, our data strongly suggest that HBV-associated ALF is mediated by a T cell-independent intrahepatic B-cell response against the core antigen of HBV that is associated with complement-mediated massive hepatocellular damage. 2. Molecular mechanisms of pathogenesis of chronic liver diseases Cirrhosis develops in 30-40% of patients with chronic hepatitis B and C, but up to 80% in those with chronic hepatitis D. It may be a stable disease for decades or it may lead to liver-related death for decompensation or HCC. The biological reasons why some patients die of liver cirrhosis complications and some do not are presently unknown. However, this variable clinical outcome suggests that not all cirrhosis is the same. To investigate these important questions, we started extensive transcriptional studies in patients with end-stage liver cirrhosis of different etiology (HBV, HCV, HCV plus alcohol, HDV, alcohol, and autoimmune), as well in normal liver donors. We studied 74 normal livers not only to build a control group for comparison with liver disease but also to study why an older age (>40) at the time of HCV infection and the male gender are associated with a worse outcome. Interestingly, we found genes differentially expressed according to age and gender, and we are now determining whether these specific gene signatures are associated with innate or adaptive immune responses, increased fibrinogenesis, or decreased fibrinolysis. Overall, 275 liver specimens from patients who underwent orthotopic liver transplantation for end-stage liver cirrhosis were processed by microarray. Our data show a distinct gene signature for each type of cirrhosis, with a striking and unexpected heterogeneity even between biologically related conditions such as HBV and HDV cirrhosis. A thorough characterization of genes associated with liver cirrhosis will be critical to identify which genes are involved in the progression of cirrhosis toward HCC. 3. Pathogenesis of HCC and search for biomarkers for the early detection of HCC The major etiologic agents of HCC have been identified but the mechanisms of hepatocarcinogenesis are still unknown. In particular, there is limited information on the alterations present in the nontumor tissue that surrounds the tumor. Because of the difficulty in studying sequential liver samples from the same patient progressing toward HCC, we have decided to map the tumor lesion as well as the unaffected areas of the liver to see whether specific alterations in gene expression are also present in the neighboring tissue. In collaboration with Dr. Zamboni, we have designed a protocol that includes 5 biopsies from the tumor, 1 from the center (A) and 4 (North, South, East, West) from the periphery (B), and 12 outside the tumor, 4 at 1 cm (C), 4 at 3 cm (D) and 4 at the edge of the liver (E). We have so far analyzed by microarray 462 liver specimens from patients with HCC of different etiology. Remarkably, we found a different molecular signature for each tumor, but even more important differentially expressed genes showed a distinct gradient of expression from non-HCC cirrhosis to tumor-surrounding tissues to tumor tissues. Strikingly, in HCV-related HCC, we found that selected genes differentially expressed in the tumor were also altered in nontumoral surrounding tissues starting from the most distant sites. The identification of gene expression alterations in nontumoral liver tissue surrounding HCC may shed light on the multistep process of hepatocarcinogenesis and provide new diagnostic or prognostic markers for HCC. 4. Correlation between viral evolution and clinical outcome in HCV infection The early evolution of the HCV quasispecies was shown to predict the outcome of acute hepatitis C (Farci et. al Science 2000), but little is known on whether the pattern of viral evolution in progressing hepatitis differs between slow and rapid progressors. Six patients were selected to represent two different clinical outcomes: 3 were slow progressors with a stable disease for >20 yrs and 3 had a rapidly progressive disease leading to liver-related death within 5 to 10 years from the onset of the infection. HCV quasispecies was studied from the first PCR-positive sample, within 2 weeks of infection, for up to 23 years. A total of 1793 sequences from the E1 and E2 genes, including the hypervariable region 1, were analyzed with newly refined bioinformatic tools. Our study provides evidence for a genetic bottleneck during the early phase of chronic HCV infection, showing a correlation between persistence of pre-existing strains after the bottleneck and rapid disease progression. Thus, the effectiveness of the host immunologic control may critically affect the pace of disease progression. 5. Search for new hepatitis agents PBC is generally thought to be an autoimmune disease though an infectious agent has not been excluded. We have identified a Sardinian patient with early PBC (stage 1). The aim of this study was to attempt to transmit an infectious agent from PBC to chimpanzees. We are currently following a chimpanzee inoculated with serum from the patient with PBC (week 104 post-inoculation). We have examined all weekly liver biopsies by microarray and we are now analyzing the data to identify specific genes related to innate immunity or mitochondria or other cellular genes. If there is evidence of an infectious agent, we will attempt to identify the putative agent by molecular techniques.